Immunocytochemistry for death cells - Houston, I have a problem (Nov/10/2009 )
I'm working with cell lines and death-inducers. I tried some immunofluorescence assays, but I'm with a problem.
First of all, let me tell you what I usually do. I grow cells in coverslips inside 6-well plates. Then, I incubate the cells with medium treated with death-inducing drugs and then I do the immunofluorescence. The first step of my protocol consists in discarding the medium and wash the cells with PBS. However, death cells dettach from the plate/coverslips and, consequently, they are thrown off. When I finish the procedure I have a small representation of death cells.
I could do cytospins for the medium with death-cells, but, in this case I would only get death cells in the assay...
What can I do? This week I remembered something: gather the medium with death-cells, trypsinize the attached cells and do all the procedure in suspension and, in the end, do a cytospin. However, I loose a lot of death-cells in centrifugations...
What should I do?
When I perform this kind of experiments normally I am very carefully with the addition of PBS and normally I don't exceed too much with the time of cell death induction. I don't have huge loses of cells.
Try to do a cytospin of death cells above the coverslip used and fixe cells if you don't have better results.